WO1994026912A1 - Procede et vecteur recombine pour accroitre l'expression de transgenes - Google Patents

Procede et vecteur recombine pour accroitre l'expression de transgenes Download PDF

Info

Publication number
WO1994026912A1
WO1994026912A1 PCT/EP1994/001408 EP9401408W WO9426912A1 WO 1994026912 A1 WO1994026912 A1 WO 1994026912A1 EP 9401408 W EP9401408 W EP 9401408W WO 9426912 A1 WO9426912 A1 WO 9426912A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
promoter
sequence
rna
vector constructs
Prior art date
Application number
PCT/EP1994/001408
Other languages
German (de)
English (en)
Inventor
Robert-Matthias Leiser
Lutz Plobner
Klaus MÜNTZ
Original Assignee
Institut für Pflanzengenetik und Kulturpflanzenforschung
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut für Pflanzengenetik und Kulturpflanzenforschung filed Critical Institut für Pflanzengenetik und Kulturpflanzenforschung
Priority to AU68423/94A priority Critical patent/AU6842394A/en
Publication of WO1994026912A1 publication Critical patent/WO1994026912A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation

Definitions

  • the subject of this patent is a method and the vector constructs used therefor for regulating the expression of transgenes in recipient organisms, preferably plant recipients.
  • the method is based on the use of the replication mechanism of RNA viruses for the propagation of desired messenger RNA at the post-transcriptional level.
  • the foreign gene is inserted into a construct according to the invention which additionally codes for a viral replicase or corresponds to this construct with a further construct, these constructs being under the transcriptional control of a constitutive or tissue-specific or ontogenesis-specific promoter and at least some of the resulting ones Transcripts are recognized by the virus replicase as a template like the homologous virus RNA.
  • the constructs are introduced into the recipient, possibly integrated into the recipient genome and transcribed using recipient's own transcription systems.
  • Expression optimization is achieved in that the (a) transcript is initially used as a messenger for the Synthesis of virus replicase is used. This in turn recognizes at least the transcript coding for the transgene of interest as a template for a preferably recurring replication, in the course of which there is a post-transcriptional increase in the messenger of interest and finally the translation of the desired transgenic sequence, provided the transgene is a protein-coding sequence .
  • transgenic organisms crucially depends on whether and how it is possible to regulate the expression of the desired transgene.
  • the expression level is very often very low.
  • a system is therefore required with which an increase in expression can be achieved to a much greater extent than with the previous (above) experimental approaches, whereby at the same time it must be attempted that this system can evade certain regulatory principles of the normal functional apparatus of the cell.
  • This can be achieved by integrating the transgene into the genome of a suitable virus and treating it like a virus gene in the infected cell during the replication process.
  • the crucial disadvantage of this approach is that this expression system cannot be made stable. The recipient organism must first be infected with the chimeric virus. Here one encounters limits that result from the host circle of the virus and its preferred replication sites in the recipient.
  • the method according to the invention and the constructs used therefor can be used wherever there is a significant increase in the amount of gene product of the transgene, e.g. for projects of "protein farming” or for shifting the composition of a total protein of a gene family in favor of the transgene product, as is the case with the genetic engineering improvement of the amino acid composition of seed storage proteins in plants.
  • a solution is achieved with which it is possible to control the expression level of a transferred gene in the recipient organism, preferably to increase it.
  • This increase in the expression level is significantly higher than is achieved in other previously known methods.
  • the patented method is based on the use of the replication mechanism of RNA viruses to multiply desired messenger RNA at the post-transcriptional level.
  • the desired sequences from the genes to be transferred are inserted into the respectively suitable patented vector constructs and transferred in this form into the recipient organism in a suitable manner (e.g. by Agrobacterium-mediated or biolistic gene transfer). The method can depend on
  • the goal should be designed so that the gene transfer strives for stable integration in the nuclear or organelle genome or transient expression with appropriate transcription control (based on constitutive or regulatable promoters).
  • the vector constructs according to the invention are based on the genome of RNA viruses, preferably plant viruses, and in particular contain at least part of the viral replica complex.
  • the integration of the foreign sequence is designed according to the invention in such a way that the primary transcript formed after transfer in vivo has recognition sequences which make it possible for the primary transcript to be recognized and treated by the virus replicase like the virus RNA or a part of it itself.
  • Foreign sequence and virus sequence can, however, preferably not necessarily be placed on one and the same construct. As a result, the transfer and ultimately the transcription of both parts of the patented construct can also take place separately.
  • the virus genomes or parts of them are incorporated into constructs via reverse genetics, which provide a suitable promoter and - if necessary - the polyadenylation signal.
  • the promoters are either constitutive, e.g. the 35S promoter of the cauliflower mosaic virus, or tissue- or ontogenesis-specific promoters, e.g. seed-specific promoters.
  • the linkage of promoter and DNA copy of the virus genome is designed so that the start of transcription largely coincides with the start of the virus genome, i.e. that the later transcript largely corresponds to the 5'-terminus of the viral RNA, if possible without extranucleotides, which could negatively influence the desired recognition of the RNA by the viral replicase.
  • the virus genome is preferably modified in such a way that all virus genes which are not necessary for the replication process within a primarily infected cell have been inactivated by deletion or reading frame-modifying mutagenesis. These include the
  • ERSATZBL ⁇ 1T Example those virus genes which code for the coat protein, the transport from cell to cell and / or the vector transfer. In this way it is achieved that the later chimeric constructs cannot spread over the cells that have been transfected.
  • constructs based on the genome of the beet western yellows virus (BWYV) were created and linked to various model genes, whereby the formation of the primary transcripts was controlled by both the 35S promoter (constitutive) and a seed-specific promoter.
  • the foreign gene sequences were preferably inserted instead of the coat protein gene, so that their expression took place under the control of the subgenomic promoter of the BWYV.
  • the method according to the patent can preferably be used for optimizing the expression of foreign genes, for the purpose of "improving" certain properties of the recipient organism, e.g. the amino acid composition of the seed storage tototems or resistance to various biotic or abiotic harmful factors) or to obtain foreign gene products by means of a so-called gene arming were used.
  • plant viruses as episomal vectors represents a possibility for gene expression of a completely different kind. After integration of the foreign gene in the virus genome, it is expressed like a virus gene during replication. A major advantage of such a system is that the foreign gene is amplified together with the virus genome by replication, which creates the conditions for high expression (Joshi and Joshi, 1991).
  • DNA viruses were among the first plant viruses that appeared attractive as episomal vectors.
  • CaMV cauliflower mosaic virus
  • attempts have been made to insert additional sequences at different locations in the genome or to replace existing ones. Small insertions were tolerated in several places (Hüll, 1985), there were strict limitations for longer sequence sections to be inserted.
  • DNA molecules that are too large are no longer packed in particles, and on the other hand, the exchange of virus genes for foreign genes has a drastic effect on replication or virus transport.
  • Only the open reading frames II and VII can be deleted without influencing the infectivity of these constructs (Howarth et al., 1981; Dixon and Hohn, 1984).
  • Certain mutations in open reading frame (ORF) VII are lethal or revert in vivo
  • gemini viruses Another group of DNA plant viruses that have been used as episomal expression vectors are the gemini viruses. Your genome consists of one or two molecules of single-stranded DNA. They are interesting as expression vectors above all because some gemini viruses have host plants among the monocotyledons.
  • TGMV tomato golden mosaic virus
  • the coat protein is not necessary for the replication and systemic spread of this virus and can therefore be deleted (Etessami et al., 1988; Gardiner et al., 1988; Hayes et al., 1988). Instead of the open reading frame for the coat protein, coding regions for the CAT, GUS or NPT II gene were inserted (Hanley-Bowdoin et al., 1988; Hayes et al., 1988; Hayes et al., 1989; Kanievski et al. 1992). Infection with such constructs occurred through agroinoculation (Grimsley et al., 1986).
  • the insert size of the insert did not decisively influence the replication in the TGMV, the insert size is limited in the geminivirus cassava latent virus (CLV) (Ward et al., 1988; Etessami et al., 1989).
  • CLV cassava latent virus
  • a high level of expression of the inserted foreign gene and systemic were found for recombinant CLV genomes Spreading observed in plants (Ward et al., 1988).
  • the activity of the marker enzymes expressed by replicating WSV vectors was 20 times higher than that of non-replicating control vectors. It was comparable to the values transiently achieved in cells of T. monococcum by a 35S promoter-driven NPT II gene (Matzeit et al., 1991).
  • the results for extrachromosomal amplification and expression of foreign genes with the aid of DNA virus genomes show that the exchange of viral genes for foreign sequences is possible in principle.
  • the foreign sequences are propagated together with the virus genome in plant cells and expressed exactly like the corresponding virus gene.
  • the level of expression of the inserted foreign genes is estimated to be high. Except for exceptions (e.g. Matzeit et al., 1991), exact comparisons with plant promoters have not been made.
  • the use of such methods has disadvantages, which are specific for the virus used on the one hand and are generally expressed in the case of DNA viruses on the other.
  • the CaMV host circle for example, is very small and plants outside the host circle cannot be infected with chimeric constructs.
  • the insertion size of all viruses in the Caulimovirus group is still strictly limited (maximum - 1 kb). Even within this limit, foreign sequences are deleted relatively frequently, which is due to the retroviral replication mechanism of these viruses (Grimsley et al., 1986). Virus genomes with an integrated foreign gene remain infectious and induce symptoms in the host plant.
  • the exchange of the ORF II of the CaMV for a foreign gene only prevents the transmission by aphids. Cloned DNA is also mechanically transferable (Fütterer et al., 1990).
  • TGMV gemini viruses with a two-part genome
  • one part of the genome is necessary for the systemic spread in plants. If the coat protein gene, which is located on the other part of the genome, is replaced by a foreign gene, the second component must be brought into the plant in order to achieve a high level of expression through systemic spread.
  • One way to avoid such double infections is the stable integration of the foreign component as a tandem copy in the genome of the plant (Hayes et al., 1988a; Kanievski et al., 1992).
  • the exchange of the coat protein gene for gemini viruses with a one-part genome is in any case with one Loss of ability to systemic spread associated. Application is therefore limited to plant cell cultures (Matzeit et al., 1991).
  • the size of the insert is less limited for gemini viruses than for caulimoviruses, but there are conflicting results about the stability of large insertions (Stanley and Townsend, 1986; Ward et al., 1988).
  • the stable integration of tandem copies in the plant genome would be a way out to reduce the effect of instability that might occur (Davies and Stanley, 1989).
  • RNA viruses replicate via a virus-coded RNA-dependent RNA polymerase (replicase). The enzyme is specific for the corresponding virus and ensures a high rate of reproduction. The resulting daughter RNA molecules with (+) strand polarity can act directly as messenger RNA in some viruses.
  • RNA RNA that is a polyprotein is formed, which is later limited, site-specific proteolysis is broken down into the individual components.
  • the expression of the 3 '-related genes takes place via the formation of subgenomeric RNA from the (-) - strand intermediate of the genomic RNA.
  • the subgenomic RNA is active as messenger RNA.
  • the coat protein genes in some plant viruses are expressed via subgenomic RNA. In view of the high level of expression, it was above all the sugenomic promoters for the envelope protein genes which were to be used for controlling the expression of foreign genes.
  • the brome mosaic virus served as the vector.
  • This virus has a three-part RNA genome. Two parts of the genome code for replication functions that also increase RNA3 in trans, on which there are 2 open reading frames (Ahlquist et al., 1981).
  • a subgenome RNA is formed from the RNA3, which encodes the coat protein.
  • the BMV appeared to be particularly suitable as an episomal vector for the following reasons (French et al., 1986). Due to the divided genome and the trans efficiency of the replicase, the RNA3 is available for genetic engineering manipulation without negatively influencing the replication process as such. Provided that the replicase recognizes the altered RNA3, this is increased including the inserted foreign gene.
  • the virus reproduces extremely strongly in host plant cells. In infected plants, the coat protein accumulates in milligram amounts per gram of tissue.
  • RNA3 modified in vitro transcripts of the RNA3 were inoculated together with RNAl and 2 in protoplasts (Hordeum vulgare). In terms of molar ratio, approximately half as much truncated subgenome RNA was formed as when inoculated with wild-type RNA. In other constructs, the deleted region was replaced by a CAT gene. The chimeric RNA accumulated 5-15 times weaker than wild-type RNA. However, the CAT enzyme activity, which was measured in inoculated protoplasts, was extraordinarily high. In comparison to transgenic plants with expression of the CAT gene under the control of a Rubisco or Nos promoter (Herrera-Estrella et al., 1984), the measured activity was 7 and 23 times as high, respectively.
  • Jupin et al. (1990) searched for cis-active elements that are important for the replication of the RNA3 of the beet necrotic yellow vein virus (BNYW).
  • BNYW beet necrotic yellow vein virus
  • the virus consists of several (4) parts of the genome, of which only the RNAl and 2 are required for replication in plants. Approximately 75% of the internal area of the RNA3 could be deleted without losing the replicability of the RNA3.
  • the GUS gene was inserted instead of the existing reading frame. Such transcripts of such cDNA mutants were propagated after co-inoculation with RNAl and 2 in Chenopodium quinoa. High GUS activity has been demonstrated in leaves of such plants. It is therefore possible to create a modified replicon.
  • RNA3 and RNA4 are variable components of the virus. Depending on the type of transmission and the propagation host, these RNAs can carry deletions or disappear completely from the isolates (Koenig et al., 1986). Due to the lack of selection, it will therefore hardly be possible to stabilize the foreign gene over a long period of time.
  • RNA viruses with a shared RNA genome the barley stripe mosaic virus (BSMV) has also been used as an episomal expression vector for foreign genes (Joshi et al., 1990).
  • the coat protein gene of the RNAß was exchanged for the luciferase gene and transcripts were transfected together with transcripts of the RNAa and g in protoplasts.
  • a 42-fold higher enzyme activity was measured in tobacco protoplasts.
  • the activity increased by Virus amplification 60 hours after transfection even 123 times.
  • TMV Tobacco Mosaic Virus
  • RNA was detectable (genomic and subgenomic) than in parallel experiments with the wild-type RNA or the mutant without a coat protein.
  • the lack of coat protein which plays a role in long-distance transport in plants (Atabekov and Dorokhov, 1984), can be attributed to the fact that CAT activity was not systemically detectable.
  • the CAT activity in the inoculated leaf tissue measured in these experiments was lower than was to be expected from the coat protein expression. This is attributed to the weak replication of the chimeras.
  • Dawson et al. (1989) constructed a hybrid virus in which a CAT gene was inserted into a complete TMV genome under the control of a duplicate of the subgenomic coat protein gene promoter. The additional gene was either in front of or behind the coat protein gene. In this way it was achieved that the chimeric RNA was packed into virus particles.
  • the hybrid virus with the CAT gene insertion before the coat protein ORF replicated in tobacco like wild-type TMV. It spread systemically and produced weak mosaic symptoms in young leaves. There was high CAT activity in inoculated leaves. However, little or no enzyme activity could be detected in systemically infected leaves. Low activity correlated with a decrease in the concentration of subgenomic RNA in Northern blots. After prolonged infection, the wild-type RNA was reconstituted. The latter is attributed to homologous recombination on duplicated sequence sections (subgenomic promoter).
  • Insertion of the CAT gene immediately before the 3 'end of the virus RNA had a drastic effect on the replication of this hybrid virus in plants. This resulted in poor symptom expression and low expression of the CAT gene.
  • Transient expression systems based on virus genomes ie without integration of the desired foreign gene in the plant genome, have advantages over the usual stable expression methods. Since it is possible to transfect protoplasts, cell cultures or intact plants with virus - RNA transcripts or derivatives with high efficiency, there is an advantage in that sometimes lengthy and sometimes difficult regeneration processes can be avoided. Some RNA viruses (e.g. the TMV) also have an extremely broad host spectrum, which makes many plant species accessible for such manipulations. In the event of the unrestricted spread of the transfected constructs, plants can be used at any stage of development and harvested after reaching the maximum level of expression of the foreign protein.
  • the RNA replicates with the help of the specially coded replicase and from host components up to a high number of copies per cell.
  • the use of viral subgenomic promoters for the initiation of the synthesis of foreign gene mRNA creates a large number of matrices for the foreign protein synthesis.
  • the coat protein is usually the most frequently synthesized virus protein. It can be isolated in milligram quantities from one gram of fresh mass. If foreign proteins can be produced with similar efficiency by plant cells, the level of expression is orders of magnitude higher than the level that can be achieved by strong plant promoters.
  • the expression is also independent of so-called position effects. The latter is a phenomenon in the case of foreign genes which are stably integrated in the genome and which makes it necessary to generate and test many independent transformants in order finally to select those with high foreign gene expression.
  • RNA virus systems are autonomous. If the RNA gets into the plant cell, the replication can no longer be controlled. On the one hand, tissue-specific expression is excluded. The foreign protein is created where the virus can also replicate. On the other hand, this raises safety concerns. Because together with easy transferability of the RNA (mechanically) to other plants, such constructs could get out of control.
  • RNA mechanically
  • RNA replicases have no proof reading mechanisms, which leads to errors after a few replication cycles, particularly in the insert, because the selection is missing here.
  • large parts or the entire inserted sequence can be deleted by recombination, in particular on duplicated viral sequences, in a relatively short time. The deletion of the insert quickly leads to the cessation of the foreign protein synthesis because the resulting RNA species replicate better and compete effectively with insert-bearing forms.
  • virus-encoded functions can be stably integrated and expressed as chimeric genes in the genome of target plants. It is known from studies of virus transport that genes for transport proteins expressed in transgenic plants complement defects in the virus genome (Deom et al., 1987; van Dun et al., 1988). Gene exchange mutants of RNA viruses in which a foreign gene has been replaced by a transport protein could be complemented in the same way (Joshi and Joshi, 1991). "Containment" conditions can be created in this way, since the foreign-bearing virus construct can only spread in corresponding transgenic plants. The same strategy was followed for replicase genes (van Dun et al., 1988) and for coat protein genes.
  • RNA viruses with a shared genome seem to be predestined for this purpose (BMV, BNYW and others).
  • the replicases of these viruses act naturally in trans, ie they amplify not only the RNA genomes on which they are encoded but also others.
  • the regulatory sequences for the amplification are largely limited to the immediate 5 'and 3' ends of the RNA to be amplified.
  • a full-length clone of the virus was constructed from cDNA fragments of different lengths, which in total partially overlap the entire BWYV sequence (see Veidt et al., 1992).
  • the clone enabled the synthesis of (+) RNA under the control of the bacteriophage T7-RNA polymerase promoter.
  • the in vitro transcripts replicated like virus RNA after electroporation in protoplasts of Chenopodium quinoa. The synthesis of the coat protein has been demonstrated.
  • the cDNA was at the 5 'end with the 35S promoter
  • ERS ⁇ ZBL of the CaMV and at the 3 'end connected to the nos polyadenylation signal.
  • a ribozyme sequence was inserted between the 3 'end of the virus sequence and the polyadenylation signal, which after the transcription autocatalytically creates the exact 3' end of the virus RNA (for methodical details, see Leiser et al., 1992).
  • an expression cassette was created which made it possible to produce BWYV-RNA in plants or plant cells via electroporation or agro-inoculation. Such in vivo transcripts replicated just like virus RNA.
  • the example explains how viral genes that are necessary for replication can be switched off by in vitro mutagenesis. At the same time, insertion sites are to be created, in which foreign sequences are then integrated.
  • the replication of such constructs and the amount of synthesis of the desired foreign gene products can be checked after transfer to protoplasts and transient expression. It should be noted that compared to conventional expression cassettes, in which the transcription of the genes is controlled exclusively by a CaMV-35S promoter, with the constructs described, depending on the experiment, between 3 and 10 times more Protein is synthesized. This is due to the fact that replication-active RNA is formed during the transcription. The reading frames for the virus-coded parts of the replicase are first translated from the RNA.
  • the RNA is replicated. This creates both Full length RNA molecules as well as subgenomic RNA.
  • the inserted reading frame for the foreign gene can be translated from the subgenomic RNA.
  • the translation is therefore preceded by an RNA multiplication, which is carried out by the viral replication mechanism of the BWYV. A large amount of mRNA is produced, which codes for the foreign proteins.
  • an Eco RV fragment and a Barn HI fragment are subcloned accordingly in the phagemids pBluescript (+) and pBS (+), respectively.
  • the plasmid clone GRN is digested with the restriction endonuclease EcoRV or BamHI and the resulting cleavage products with a size of 1500 bp or 1104 bp isolated.
  • the vectors pBluescript (+) and pBS (+) are also cleaved accordingly with EcoRV or BamHI and then dephosphorylated.
  • pBluescript (+) with the EcoRV- fragment and pBS (+) with the BamHI fragment from the clone GRN are ligated.
  • the subclones SK Eco RV and SK Barn HI are created accordingly.
  • the bacterial strain TG 1 (Amersham) serves as the host strain for all cloning.
  • the synthesis of single-stranded DNA is induced by infection of the phagemid-carrying bacteria with the helper phage K07.
  • the single-stranded DNA contains the "sense" strands of the subcloned DNA fragments.
  • mutagenesis with specific oligonucleotides is carried out in vitro.
  • the basis is a kit from Amersham (oligonucleotide-directed in vitro mutagenesis system) and the instructions it contains.
  • the sequence of the Eco RV fragment in the area of the start codon of the ORF I of the BWYV is changed by the mutagenesis in such a way that the restriction site Sna BI is produced instead of the AUG codon (see Fig. 2 (A)).
  • the destruction of the start codon and its surroundings should ensure that this reading frame is no longer unknown in the corresponding virus protein Function can be translated.
  • the sequence of the BamHI fragment in the region of the start codon of the ORF IV of the BWYV is to be converted into the Xho I restriction site. This restriction site will later serve as an insertion site for foreign sequences.
  • Muta ORF I corresponds to the clone GRN except for the mutated start codon for reading frame I. Although reading frame I can no longer be translated, the RNA of Muta ORF I transcribed in vivo in plant protoplasts is able to replicate.
  • any nucleic acid section can be inserted into the clone SK Bam HI between the newly formed Xho I site and the remaining restriction sites of the polylinker from pBS (+) downstream of the Bam HI insert.
  • the virus sequence is exchanged for the section to be inserted. It is important that after insertion at the 3 'end of the inserted fragment a Bam HI and an Eco Rl location is available.
  • the Eco Rl site is required to restore the Xho I site in the subcloned Bam HI fragment, which was previously destroyed by the filling reaction and religation. This is done by exchanging the Eco ⁇ -I fragment between the Eco Rl site (pos. 6419 in Fig. 1) on the Bam HI fragment and the required Eco Rl site on the 3 'side of the inserted nucleic acid section for the Eco RI Fragment in the clone SK Bam HI.
  • the entire insert in pBS (+) is cut out with Bam HI (a Bam HI site downstream of the foreign gene is an essential prerequisite for this).
  • the insert consists of the virus cDNA sequence from positions 2902 to 3479 (see Veidt et al., 1988) and the reading frame of the inserted gene. Virus sequence and foreign gene are linked via> the Xhol site created by mutagenesis.
  • the Bam HI fragment is isolated and exchanged for the Bam HI fragment from the clone Muta ORF I.
  • the resulting recombinant plasmid contains a foreign nucleic acid section instead of the reading frame for the coat protein from items 3480 to 4005 (see Veidt et al., 1988), which is expressed like the coat protein reading frame after RNA replication.
  • the reading frames of the ⁇ -glucuronidase and the methionine-rich 2S seed protein from Bertholetia excelsa are inserted as foreign nucleic acid segments into the construct Muta ORF I in the manner described above. It is important that the reading frames contain their own start and stop codons, which enable the correct translation into functional proteins.
  • the promoter and the polyadenylation signal of the genes are not required and are therefore deleted.
  • the resulting constructs were called GRN-GUS and GRN-BNG.
  • the plasmids are purified and transfected as DNA into plant protoplasts. The transfection done by electroporation. 15 mg of plasmid DNA are transfected into 2 x 105 protoplasts. The method for electroperation by Veidt et al. (1992) is used. After the transfection, the protoplasts are incubated in a minimal medium according to Rottier et al. (1979) for three days. After the protoplasts have been harvested, the expression analysis is carried out at the RNA and protein levels.
  • the aim of this example is to create constructs which allow stable integration in the plant genome and constitutive expression of a mutated BWYV cDNA with an integrated foreign gene.
  • the plasmids GRN-GUS and GRN-BNG from Example 1 contain individual restriction sites for the enzymes Kpn I and Sal I. By restriction digestion with both enzymes, a fragment is released in each of the plasmids, which fragment mutated from a 35S promoter of the CaMV BWYV cDNA with integrated foreign gene and a nos polyadenylation signal. These functional expression cassettes are cloned individually into the binary vector pBIN19, which has compatible restriction sites.
  • the T-DNA from pBIN 19, which contains the aforementioned exposure cassette can be integrated into the genome of plant cells using conventional methods of Agrobacterium-mediated gene transfer.
  • the Agrobacterium strains used (for example A. tumefaciens 2260 or EHA 101 for the transformation of tobacco and EHA 101 for the transformation of Vicia narbonensis) contain a resident plasmid on which the virulence functions are contained. After selection of the agrobacterial colonies that form the binary vector construct hold, plant cells are transformed with the bacterial suspensions. The leaf disc method is used to transform tobacco (N. tabacum cv. Samsun) (see Horsch et al., 1985). Vicia narbonensis is transformed by a method of Pickert et al. (). After the transformation, transgenic tissues, shoots and regenerated plants with kanamycin are selected in the medium. A corresponding resistance gene is located between the T-DNA borders in the binary vector pBIN19 and is integrated in the transformation together with the chimeric construct in the genome of the plants.
  • the modified virus RNA is transcribed and amplified by the 35S promoter (see Example 1). Integration in the genome causes the replication-active RNA to be constitutively expressed in every cell in which the 35S promoter is active.
  • the foreign proteins GUS or 2S seed protein
  • the method according to the patent has the advantage that foreign genes can be expressed in a tissue-specific or development-specific manner.
  • a seed-specific expression of the vector constructs in transgenic plants is described.
  • the mutated virus cDNA is placed under the control of a seed-specific promoter.
  • the complete promoter of a seed storage protein (phaseolin promoter) cloned in pBluescript KS (+) and the subcloned and mutated EcoRV fragment (see Example 1) form the starting point for the exact ligation of the transcription start site of the promoter with the 5 'terminus of Virus cDNA.
  • the promoter is cloned so that it is at the 3 'end an Eco RV site is located.
  • the plasmid is linearized with the enzyme Eco RV and the EcoRV fragment from the mutant clone Sk EcoRV is inserted by ligation.
  • a clone in which the reading direction of the promoter matches the corresponding sense direction of the 5 'end of the virus cDNA is selected for further processing.
  • the start of transcription of the phaseolin promoter and the 5 'end of the virus cDNA are both parts of the untranslated region of the phaseolin gene which originate from the promoter clone and 3' terminal parts of the 35S -Promotors from the individual Eco RV site (pos. 3291 in Fig. 1) to the start of transcription, which are present on the subcloned Eco RV fragment.
  • a large fragment of the plasmid is amplified by means of a PCR reaction, which, apart from the sequence to be deleted, contains the entire plasmid including the vector sequence.
  • the primers used contain at the 3 'ends a recognition sequence for the restriction endonuclease BamHI, so that the ends of the amplified fragment can be cut with this enzyme.
  • the resulting compatible ends are ligated together.
  • the resulting plasmid contains an insert from a phaseolin promoter and 5 'end of the BWYV cDNA, which are connected to one another via 6 base pairs (Bam HI site).
  • the 6 additional nucleotides are deleted with the aid of in vitro mutagenesis, thus creating the exact connection between the transcription start of the promoter and the 5 'end of the virus cDNA.
  • the resulting plasmid is digested with Eco RV and Sal I.
  • a fragment can be separated from the clones GRN-GUS and GRN-BNG with the same enzymes, the BWYV cDNA (apart from the 5 'end to the Eco RV site) including the inserted GUS or 2S seed protein gene and contains the nos polyadenylation signal.
  • This fragment is in the Eco RV / Sal I-cut plasmid ligated.
  • An expression cassette consisting of a phaseolin promoter, mutated BWYV cDNA with an integrated foreign gene (GUS or seed storage protein gene) is produced under the control of the subgenomic viral promoter and a nos polyadenylation signal.
  • the expression cassette can be cut out via the individual restriction sites for Xba I and Kpn I and cloned into a binary vector (eg pGA 482).
  • a binary vector eg pGA 482.
  • REPLACEMENT BLADE L. Herrera-Estrella, G. van den Broeck, R. Maenhaut, M. van Montagu, J. Schell, M. Timko, A. Cashmore (1984): Light-- inducible and chloroplast- associated expression of a chimeric gene introduced into Nicotiana tabacum using a Ti-plasmid vector. Nature, 310, pp. 115-120
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE YES
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 2: GTTATCCTGA GTACGTAATT GATCACCTAA GG 32
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE YES
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

En vue d'accroître l'expression de transgènes, le procédé selon l'invention consiste à transférer au moins une séquence spécifique de nucléotides, après insertion dans le vecteur recombiné, dans l'organisme-receveur, de préférence eucaryote, et à transcrire au moins une partie de cette séquence de nucléotides, les transcripts ainsi formés étant amplifiés par un système de réplication d'ARN, de préférence d'origine phytovirale.
PCT/EP1994/001408 1993-05-06 1994-05-04 Procede et vecteur recombine pour accroitre l'expression de transgenes WO1994026912A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU68423/94A AU6842394A (en) 1993-05-06 1994-05-04 Method and vector construct for increasing the expression of transgenes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4315109.4 1993-05-06
DE19934315109 DE4315109A1 (de) 1993-05-06 1993-05-06 Verfahren und Vektorkonstrukt zur Expressionssteigerung von Transgenen

Publications (1)

Publication Number Publication Date
WO1994026912A1 true WO1994026912A1 (fr) 1994-11-24

Family

ID=6487402

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1994/001408 WO1994026912A1 (fr) 1993-05-06 1994-05-04 Procede et vecteur recombine pour accroitre l'expression de transgenes

Country Status (3)

Country Link
AU (1) AU6842394A (fr)
DE (1) DE4315109A1 (fr)
WO (1) WO1994026912A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998055636A1 (fr) * 1997-06-02 1998-12-10 Joseph Atabekov Construction de recombinaison permettant d'ameliorer l'expression genique chez les plantes
US6342372B1 (en) 1993-09-15 2002-01-29 Chiron Corporation Eukaryotic layered vector initiation systems for production of recombinant proteins
US6767699B2 (en) 2000-05-31 2004-07-27 Chiron Corporation Method for the quantitation of alphavirus replicon particles
US7811812B2 (en) 1996-04-05 2010-10-12 Novartis Vaccines & Diagnostics, Inc. Recombinant alphavirus-based vectors with reduced inhibition of cellular macromolecular synthesis
US8647864B2 (en) 1999-04-14 2014-02-11 Novartis Ag Compositions and methods for generating an immune response utilizing alphavirus-based vector systems
KR20210075708A (ko) * 2019-12-13 2021-06-23 대한민국(농촌진흥청장) 박과진딧물매개황화바이러스 감염성 클론 및 이의 용도

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0298918A2 (fr) * 1987-07-10 1989-01-11 Ciba-Geigy Ag Résistance inductible contre des virus dans des plantes
CN1033645A (zh) * 1988-10-22 1989-07-05 中国科学院上海植物生理研究所 控制植物病毒病的基因工程方法
WO1989008145A1 (fr) * 1988-02-26 1989-09-08 Biosource Genetics Corporation Transformation chromosomique non nucleaire
EP0412006A1 (fr) * 1989-08-04 1991-02-06 Plant Genetic Systems, N.V. Plantes à fleurs, semences ou embryons modifiés
EP0425004A2 (fr) * 1989-10-03 1991-05-02 Aveve N.V. Manipulations génétiques avec l'ADN recombinant, contenant des séquences dérivées des virus d'ARN
WO1991013994A1 (fr) * 1990-03-13 1991-09-19 Commonwealth Scientific And Industrial Research Organisation Expression de genes
EP0479180A2 (fr) * 1990-10-05 1992-04-08 Hoechst Aktiengesellschaft Plantes résistantes aux virus, procédé pour leur production
EP0573767A1 (fr) * 1992-04-28 1993-12-15 Nihon Nohyaku Co., Ltd. Procédé de préparation d'un gène étranger ou son produit dans les cellules de plante
WO1994016089A1 (fr) * 1992-12-30 1994-07-21 Biosource Genetics Corporation Amplification virale de l'arn messager recombine dans les plantes transgeniques

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0298918A2 (fr) * 1987-07-10 1989-01-11 Ciba-Geigy Ag Résistance inductible contre des virus dans des plantes
WO1989008145A1 (fr) * 1988-02-26 1989-09-08 Biosource Genetics Corporation Transformation chromosomique non nucleaire
CN1033645A (zh) * 1988-10-22 1989-07-05 中国科学院上海植物生理研究所 控制植物病毒病的基因工程方法
EP0412006A1 (fr) * 1989-08-04 1991-02-06 Plant Genetic Systems, N.V. Plantes à fleurs, semences ou embryons modifiés
EP0425004A2 (fr) * 1989-10-03 1991-05-02 Aveve N.V. Manipulations génétiques avec l'ADN recombinant, contenant des séquences dérivées des virus d'ARN
WO1991013994A1 (fr) * 1990-03-13 1991-09-19 Commonwealth Scientific And Industrial Research Organisation Expression de genes
EP0479180A2 (fr) * 1990-10-05 1992-04-08 Hoechst Aktiengesellschaft Plantes résistantes aux virus, procédé pour leur production
EP0573767A1 (fr) * 1992-04-28 1993-12-15 Nihon Nohyaku Co., Ltd. Procédé de préparation d'un gène étranger ou son produit dans les cellules de plante
WO1994016089A1 (fr) * 1992-12-30 1994-07-21 Biosource Genetics Corporation Amplification virale de l'arn messager recombine dans les plantes transgeniques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 113, no. 14, 1990, Columbus, Ohio, US; abstract no. 122723, JUN, W.: "Preparation of transgenic plants for control of virosis" *
LEISER, R.-M., ET AL.: "Agroinfection sa an alternative to insects for infecting plants with beet western yellows luteovirus", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 89, October 1992 (1992-10-01), WASHINGTON US, pages 9136 - 9140 *
YOUNG, M., ET AL.: "Using plant virus and related RNA sequences to control gene expression", STADLER GENETICS SYMPOSIA SERIES: GENE MANIPULATION IN PLANT IMPROVEMENT, II, 1990, pages 313 - 330 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6342372B1 (en) 1993-09-15 2002-01-29 Chiron Corporation Eukaryotic layered vector initiation systems for production of recombinant proteins
US6376236B1 (en) 1993-09-15 2002-04-23 Chiron Corporation Recombinant alphavirus particles
US7572628B2 (en) 1993-09-15 2009-08-11 Novartis Vaccines And Diagnostics, Inc. Eukaryotic layered vector initiation systems
US7977091B2 (en) 1993-09-15 2011-07-12 Novartis Vaccines & Diagnostics, Inc. Eukaryotic layered vector initiation systems
US7811812B2 (en) 1996-04-05 2010-10-12 Novartis Vaccines & Diagnostics, Inc. Recombinant alphavirus-based vectors with reduced inhibition of cellular macromolecular synthesis
WO1998055636A1 (fr) * 1997-06-02 1998-12-10 Joseph Atabekov Construction de recombinaison permettant d'ameliorer l'expression genique chez les plantes
US6573427B1 (en) 1997-06-02 2003-06-03 Joseph Atabekov Recombinant construct for enhancement of gene expression in plants
US8647864B2 (en) 1999-04-14 2014-02-11 Novartis Ag Compositions and methods for generating an immune response utilizing alphavirus-based vector systems
US6767699B2 (en) 2000-05-31 2004-07-27 Chiron Corporation Method for the quantitation of alphavirus replicon particles
KR20210075708A (ko) * 2019-12-13 2021-06-23 대한민국(농촌진흥청장) 박과진딧물매개황화바이러스 감염성 클론 및 이의 용도
KR102342405B1 (ko) 2019-12-13 2021-12-24 대한민국 박과진딧물매개황화바이러스 감염성 클론 및 이의 용도

Also Published As

Publication number Publication date
DE4315109A1 (de) 1994-11-10
AU6842394A (en) 1994-12-12

Similar Documents

Publication Publication Date Title
DE69924484T2 (de) Dsrna-bedingte regulation der gen-expression in pflanzen
DE68929521T2 (de) Nicht-nukleare chromosomale Transformation
EP1294907B1 (fr) Vecteurs binaires pour transformation amelioree de systemes vegetaux
JP5745857B2 (ja) タンパク質発現系
DE69834715T2 (de) Verfahren zur genetischer abschaltung in transgenpflanzen
DE69834908T2 (de) Verfahren zur erhöhung der expression von endogenen oder exogenen genprodukten in pflanzen
DE69831265T2 (de) Zusammensetzungen und verfahren für die genetische modifikation von pflanzen
DE69835209T2 (de) Regulatorische sequenzen für transgene pflanzen
DE10121283B4 (de) Verfahren und Vektoren zur Amplifikation oder Expression von gewünschten Nucleinsäuresequenzen in Pflanzen
DE69533037T2 (de) Pflanzentraskriptionsregulator von circovirus
DE69333267T2 (de) Erhöhte expression in pflanzen
DE10109354A9 (de) Rekombinante virale schaltersysteme
EP2354233B1 (fr) Promoteur synthétique pouvant être induit par un pathogène
DE10109354A1 (de) Rekombinante virale Schaltersysteme
EP1392833B1 (fr) Transformation d'un site cible faisant appel a des vecteurs d'amplification
DE10061150A1 (de) Verfahren und Vektoren zur Erzeugung von transgenen Pflanzen
US20030229920A1 (en) Gene silencing materials and methods
DE60132473T2 (de) Auf viren basierende amplifikationsvektoren für pflanzen
EP0421376B1 (fr) L'ARN multifonctionnelle avec une activité d'automaturation, sa production et utilisation
DE10129010A1 (de) Verfahren und Vektoren zur Erzeugung von transgenen Pflanzen
DE10101276A1 (de) Verfahren und Vektoren zur Transformation von Plastiden
WO1994026912A1 (fr) Procede et vecteur recombine pour accroitre l'expression de transgenes
DE60131075T2 (de) Durch freisetzung in geschlossener, zirkulärer form aus einer grösseren nukleotidsequenz charakterisiertes konstrukt, welches die ortsspezifische und/oder entwicklungsspezifische, regulierte expression selektierter, genetischer sequenzen erlaubt
EP0428881B1 (fr) ARNs à activité endonucléase et antisens, leurs production et utilisation
US20030033635A1 (en) Self-excising polynucleotides and uses thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA